Title: CAKUT and Autonomic Dysfunction Caused by Acetylcholine Receptor Mutations
Abstract: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs∗81 and p.Ser340∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT. Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs∗81 and p.Ser340∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT. Congenital anomalies of the kidney and urinary tract (CAKUT) represent up to 20%–30% of all prenatally detected anomalies and are the most common cause of chronic kidney disease in the first three decades of life.1Ingelfinger J.R. Kalantar-Zadeh K. Schaefer F. World Kidney Day Steering CommitteeWorld Kidney Day 2016: Averting the legacy of kidney disease-focus on childhood.Pediatr. Nephrol. 2016; 31: 343-348Crossref PubMed Scopus (14) Google Scholar, 2Calderon-Margalit R. Golan E. Twig G. Leiba A. Tzur D. Afek A. Skorecki K. Vivante A. History of Childhood Kidney Disease and Risk of Adult End-Stage Renal Disease.N. Engl. J. Med. 2018; 378: 428-438Crossref PubMed Scopus (62) Google Scholar, 3Queisser-Luft A. Stolz G. Wiesel A. Schlaefer K. Spranger J. Malformations in newborn: results based on 30,940 infants and fetuses from the Mainz congenital birth defect monitoring system (1990-1998).Arch. Gynecol. Obstet. 2002; 266: 163-167Crossref PubMed Scopus (152) Google Scholar The discovery of more than 40 monogenic causes of CAKUT in humans has led to the understanding that urogenital malformations often arise from defects in the signaling pathways that regulate nephrogenesis.4van der Ven A.T. Vivante A. Hildebrandt F. Novel Insights into the Pathogenesis of Monogenic Congenital Anomalies of the Kidney and Urinary Tract.J. Am. Soc. Nephrol. 2018; 29: 36-50Crossref PubMed Scopus (47) Google Scholar, 5van der Ven A.T. Connaughton D.M. Ityel H. Mann N. Nakayama M. Chen J. Vivante A. Hwang D.Y. Schulz J. Braun D.A. et al.Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.J. Am. Soc. Nephrol. 2018; 29: 2348-2361Crossref PubMed Scopus (48) Google Scholar, 6Verbitsky M. Westland R. Perez A. Kiryluk K. Liu Q. Krithivasan P. Mitrotti A. Fasel D.A. Batourina E. Sampson M.G. et al.The copy number variation landscape of congenital anomalies of the kidney and urinary tract.Nat. Genet. 2019; 51: 117-127Crossref PubMed Scopus (41) Google Scholar In addition, animal studies have demonstrated that intrauterine obstruction to urine flow can secondarily lead to CAKUT, although the genetic etiologies and molecular pathogenesis of these processes are not well understood.7Chevalier R.L. Thornhill B.A. Forbes M.S. Kiley S.C. Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy.Pediatr. Nephrol. 2010; 25: 687-697Crossref PubMed Scopus (149) Google Scholar Nicotinic acetylcholine receptors (nAChR) are heteropentameric ligand-gated ion channels that are widely expressed in the nervous system and in certain non-neuronal tissues, such as the bladder urothelium.8Albuquerque E.X. Pereira E.F. Alkondon M. Rogers S.W. Mammalian nicotinic acetylcholine receptors: from structure to function.Physiol. Rev. 2009; 89: 73-120Crossref PubMed Scopus (1116) Google Scholar,9Beckel J.M. Kanai A. Lee S.J. de Groat W.C. Birder L.A. Expression of functional nicotinic acetylcholine receptors in rat urinary bladder epithelial cells.Am. J. Physiol. Renal Physiol. 2006; 290: F103-F110Crossref PubMed Scopus (111) Google Scholar Interestingly, mice lacking Chrna3, the gene encoding the α3 nAChR subunit, develop a prominent genitourinary phenotype, with reduced bladder contractility, megacystis, and recurrent urinary tract infections.10Xu W. Gelber S. Orr-Urtreger A. Armstrong D. Lewis R.A. Ou C.N. Patrick J. Role L. De Biasi M. Beaudet A.L. Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 5746-5751Crossref PubMed Scopus (238) Google Scholar The α3 nAChR subunit mediates fast synaptic transmission in the parasympathetic, sympathetic, and enteric ganglia and plays a critical role in modulating normal bladder function.11Fowler C.J. Griffiths D. de Groat W.C. The neural control of micturition.Nat. Rev. Neurosci. 2008; 9: 453-466Crossref PubMed Scopus (821) Google Scholar To date, only one gene involved in neuronal synaptic transmission, CHRM3 (MIM: 118494), has been implicated in lower urinary tract obstruction in humans.12Weber S. Thiele H. Mir S. Toliat M.R. Sozeri B. Reutter H. Draaken M. Ludwig M. Altmüller J. Frommolt P. et al.Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome.Am. J. Hum. Genet. 2011; 89: 668-674Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Here, we describe the discovery of biallelic mutations in CHRNA3 in three families with CAKUT and additional extra-renal dysautonomic features. Approval for human subject research was obtained from the Institutional Review Board at the respective institutions, and samples were obtained after written informed consent. The index case subject, B1717-21, is a young man who was born to consanguineous parents of Arabic descent and who presented in childhood with recurrent urinary tract infections. Renal ultrasound demonstrated bilateral hydronephrosis, a thickened bladder wall, and a large post-void residual (Figure 1A). Voiding cysturethrogram (VCUG) revealed bilateral grade 5 vesicoureteral reflux (VUR) without posterior urethral valves (not shown), and the affected individual was given a diagnosis of non-neurogenic neurogenic bladder. He developed progressive renal insufficiency, and by 19 years of age, a DMSA scan demonstrated a small, atrophic left kidney with 10% residual function (Figure 1A). He also presented to the ophthalmologist in adolescence for difficulty seeing in bright light and was found to have bilateral mydriasis with impaired pupillary constriction. Moreover, orthostatic hypotension was diagnosed on routine physical examination (Table 1). The proband's brother, B1717-22, was also noted to have an impaired pupillary light reflex. He additionally has a history of recurrent urinary tract infections, although renal ultrasound revealed normal-appearing kidneys and bladder (not shown).Table 1Recessive Mutations Identified in CHRNA3 in Three Families with CAKUTFamilyEthnic OriginGenderExon (Zygosity)Nucleotide Change; Amino Acid Change (Segregation)aSegregation is listed as (maternal allele, paternal allele) when available. If parental DNA was not available, segregation is listed as ND.gnomAD Allele FrequenciesbNone of the identified CHRNA3 mutations have been reported homozygously in gnomAD, which includes exome or genome sequencing data from 141,456 unrelated individuals.Genitourinary ManifestationsDysautonomic ManifestationsOthercOne affected individual was found to have additional genetic abnormalities that were thought to explain some of his extra-renal manifestations. GM-21 has a de novo 2q31.1–32.3 duplication which may explain his facial dysmorphisms and intellectual disability. This duplication was not shared by his sister, GM-22.B1402Macedonianfemaleintron 3 (hom)c.267+2T>G (essential splice); (m. het; p. het)0/1/246,220bilat. VUR, grade IVrecurrent VUR post ureteral reimplantationCKD (stage 2)nonenoneB1717-21Arabicmaleexon 5 (hom)c.1010_1011delCA (p.Thr337Asnfs∗81); (ND)NPnon-neurogenic neurogenic bladderbilat. VUR, grade Vbilat. hydronephrosisatrophic left kidneyCKD (stage 2)impaired pupillary light reflexorthostatic hypotensionnoneΒ1717-22Arabicmaleexon 5 (hom)c.1010_1011delCA (p.Thr337Asnfs∗81); (ND)NPrecurrent UTIsimpaired pupillary light reflexnoneGM-21Pakistanimaleexon 5 (hom)c.1019C>G (p.Ser340∗); (m. het; p. het)0/4/246,010non-neurogenic neurogenic bladderleft hydronephrosisright cystic kidneyhypospadiasimpaired pupillary light reflexflat CTG tracing in uterohypertelorismbroad nasal rootintellectual disability2q31.1-32.3 duplication (de novo)GM-22Pakistanifemaleexon 5 (hom)c.1019C>G (p.Ser340∗); (m. het; p. het)0/4/246,010voiding dysfunctionrecurrent UTIsimpaired pupillary light reflexflat CTG tracing in uteroGERD, failure to thriveAbbreviations: Bilat., bilateral; CKD, chronic kidney disease; CTG, cardiotocography; GERD, gastresophageal reflux; het, heterozygous; Hom, homozygous; m, maternal allele; ND, no data; NP, not present; p, paternal allele; UTI, urinary tract infection; VUR, vesicoureteral reflux.a Segregation is listed as (maternal allele, paternal allele) when available. If parental DNA was not available, segregation is listed as ND.b None of the identified CHRNA3 mutations have been reported homozygously in gnomAD, which includes exome or genome sequencing data from 141,456 unrelated individuals.c One affected individual was found to have additional genetic abnormalities that were thought to explain some of his extra-renal manifestations. GM-21 has a de novo 2q31.1–32.3 duplication which may explain his facial dysmorphisms and intellectual disability. This duplication was not shared by his sister, GM-22. Open table in a new tab Abbreviations: Bilat., bilateral; CKD, chronic kidney disease; CTG, cardiotocography; GERD, gastresophageal reflux; het, heterozygous; Hom, homozygous; m, maternal allele; ND, no data; NP, not present; p, paternal allele; UTI, urinary tract infection; VUR, vesicoureteral reflux. We applied whole-exome sequencing (WES) and homozygosity mapping to individual B1717-21.13Braun D.A. Lovric S. Schapiro D. Schneider R. Marquez J. Asif M. Hussain M.S. Daga A. Widmeier E. Rao J. et al.Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome.J. Clin. Invest. 2018; 128: 4313-4328Crossref PubMed Scopus (39) Google Scholar,14Hildebrandt F. Heeringa S.F. Rüschendorf F. Attanasio M. Nürnberg G. Becker C. Seelow D. Huebner N. Chernin G. Vlangos C.N. et al.A systematic approach to mapping recessive disease genes in individuals from outbred populations.PLoS Genet. 2009; 5: e1000353Crossref PubMed Scopus (120) Google Scholar Mutation calling was performed in line with proposed guidelines by clinician-scientists who had knowledge of the clinical phenotypes and pedigree structure (Figure S1).15MacArthur D.G. Manolio T.A. Dimmock D.P. Rehm H.L. Shendure J. Abecasis G.R. Adams D.R. Altman R.B. Antonarakis S.E. Ashley E.A. et al.Guidelines for investigating causality of sequence variants in human disease.Nature. 2014; 508: 469-476Crossref PubMed Scopus (799) Google Scholar We identified a homozygous truncating mutation (GenBank: NM_000743.4; c.1010_1011delCA [p.Thr337Asnfs∗81]) in exon 5 of the gene CHRNA3 (Cholinergic Receptor Nicotinic Alpha 3 Subunit), which encodes the α3 nAChR subunit. The same homozygous mutation was found in the proband's affected older brother, B1717-22 (Figure 1B, Table 1). Through the use of the on-line tool, GeneMatcher,16Sobreira N. Schiettecatte F. Boehm C. Valle D. Hamosh A. New tools for Mendelian disease gene identification: PhenoDB variant analysis module; and GeneMatcher, a web-based tool for linking investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 425-431Crossref PubMed Scopus (91) Google Scholar,17Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (586) Google Scholar we identified two siblings of Pakistani descent (GM-21 and GM-22) who also have biallelic mutations in CHRNA3 (c.1019C>G [p.Ser340∗]). GM-21 was diagnosed prenatally with hydronephrosis, and post-natal imaging revealed a dilated, cystic right kidney, left hydroureteronephrosis, and a thickened, trabeculated bladder wall (Figure 1A). He was diagnosed with non-neurogenic neurogenic bladder and was managed with clean intermittent catheterizations and subsequent vesicostomy. His younger sister, GM-22, had recurrent urinary tract infections, and VCUG demonstrated a large-capacity bladder with incomplete emptying (not shown). Ophthalmology examination for both children revealed constant miosis with pupils that did not dilate, and both siblings additionally had flat cardiotocography (CTG) tracings in utero. This was detected at 36 weeks gestational age in the older child, for which he underwent emergent cesarean section. A flat CTG tracing was noticed at 29 weeks gestational age for the younger sibling and persisted until she was delivered at full term. We subsequently queried WES data in our cohort of 380 families with CAKUT and identified one additional affected individual with biallelic CHRNA3 mutations. In individual B1402, who has bilateral VUR and hydronephrosis, we detected a homozygous essential splice site mutation (c.267+2T>G). Interestingly, this individual underwent ureteral reimplantation as a child but developed recurrence of her VUR, for which she underwent a STING procedure (Figure 1A). We did not detect any biallelic mutations in CHRNA3 in a control cohort of 419 families with either nephrotic syndrome or nephronophthisis. All three CHRNA3 mutations were confirmed via Sanger sequencing (Figure S3). A summary of the clinical characteristics of the affected individuals and the mutations identified is provided in Table 1, and a schematic of the CHRNA3 exon and protein structure with locations of the three mutations is depicted in Figure 1C. Both the p.Thr337Asnfs∗81 and p.Ser340∗ variants are predicted to lead to premature termination of the protein prior to the fourth transmembrane helix (Figure 1C). As RNA was not available from the individual with the c.267+2T>G splice mutation, we used in silico prediction tools to determine the splicing effect. Because the c.267+2T>G change occurs at an obligatory splice site, we predict that this will lead to skipping of exon 3 and an in-frame deletion of 15 amino acids in the protein's extracellular ligand-binding domain. However, it should be noted that this may not recapitulate the splicing effect in vivo. In the two families in which homozygosity mapping was available, the CHRNA3 mutations were all located within regions of homozygosity by descent (Figure S4). None of the three CHRNA3 variants were present homozygously in the large population database, gnomAD (Table 1). Exome data for each proband from the three families were also analyzed for mutations in known CAKUT genes5van der Ven A.T. Connaughton D.M. Ityel H. Mann N. Nakayama M. Chen J. Vivante A. Hwang D.Y. Schulz J. Braun D.A. et al.Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.J. Am. Soc. Nephrol. 2018; 29: 2348-2361Crossref PubMed Scopus (48) Google Scholar and no causative variants were identified. However, individual GM-21 was noted to have a de novo 25.6 Mb duplication of chromosome 2q31.1–2q32.3 that was not shared by his affected sister. This is thought to contribute to his cognitive deficits, although further studies will be required for causality to be more definitively established. In order to examine whether the identified mutations in CHRNA3 affect receptor function, we performed electrophysiology and immunofluorescence studies in HEK293 cells. The α3 nAChR subunit is known to heteropentamerize with the β4 nAChR subunit,18Skok V.I. Nicotinic acetylcholine receptors in autonomic ganglia.Auton. Neurosci. 2002; 97: 1-11Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and we first aimed to determine whether the CHRNA3 mutations affect the ability of the α3β4 nAChR to induce current after stimulation with acetylcholine. Patch-clamping experiments were performed in HEK293 cells co-transfected with wild-type CHRNB4 cDNA and either wild-type or mutant CHRNA3 cDNA. Acetylcholine induced a dose-dependent inward current in cells overexpressing the wild-type α3β4 nAChR (Figure 2A). In contrast, no current was generated in cells expressing receptors composed of the splice site, p.Thr337Asnfs81∗, or p.Ser340∗ mutant α3 subunits (Figure 2A). These data demonstrate complete loss of function for the essential splice and two truncating variants. Because the two truncating mutations (p.Thr337Asnfs∗81 and p.Ser340∗) lead to premature termination of the CHRNA3 protein prior to the fourth transmembrane helix, we hypothesized that this would disrupt membrane trafficking of the mutant receptors. We expressed GFP-tagged wild-type and mutant CHRNA3 cDNA in HEK293 cells and labeled unpermeabilized cells with an antibody to the extracellular N terminus of the α3 nAChR subunit, which is expected to bind only those proteins that are inserted into the plasma membrane (Figures 2B and 2C). We detected membrane localization of the wild-type α3 nAChR subunit in 49% of transfected cells. In contrast, only 12%–13% of transfected cells demonstrated membrane staining for the p.Thr337Asnfs∗81 and p.Ser340∗ mutants, suggesting impaired membrane trafficking (Figure 2B). There is a trend toward reduced membrane localization for the essential splice site variant, but this did not reach statistical significance. Representative immunofluorescence images are depicted in Figure 2C. Permeabilized cells, in which both extracellular and intracellular labeling is established, were utilized as controls. We here discovered by whole-exome sequencing three different homozygous loss-of-function mutations in CHRNA3 in three families with CAKUT. We demonstrate that all three mutations attenuate the ability of the α3β4 nAChR to generate current after stimulation with acetylcholine. Additionally, the two truncating mutations, p.Thr337Asnfs∗ and p.Ser340∗, impair receptor trafficking to the plasma membrane. Micturition requires coordinated stimulation of the urinary bladder and urethral sphincters by the parasympathetic, sympathetic, and somatic nervous systems (Figure S5).11Fowler C.J. Griffiths D. de Groat W.C. The neural control of micturition.Nat. Rev. Neurosci. 2008; 9: 453-466Crossref PubMed Scopus (821) Google Scholar CHRNA3 mediates fast synaptic transmission in the autonomic ganglia, and we predict that loss of CHRNA3 may result in discoordinated detrusor and urethral function. CHRNA3 is also expressed in the bladder urothelium and therefore may play additional roles in regulating bladder contraction beyond its known function in the autonomic ganglia.9Beckel J.M. Kanai A. Lee S.J. de Groat W.C. Birder L.A. Expression of functional nicotinic acetylcholine receptors in rat urinary bladder epithelial cells.Am. J. Physiol. Renal Physiol. 2006; 290: F103-F110Crossref PubMed Scopus (111) Google Scholar Of interest, all three families in our cohort developed secondary upper urinary tract malformations, such as hydronephrosis and renal cysts, consistent with the notion that obstruction to urinary flow in utero can lead to abnormalities in renal development.7Chevalier R.L. Thornhill B.A. Forbes M.S. Kiley S.C. Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy.Pediatr. Nephrol. 2010; 25: 687-697Crossref PubMed Scopus (149) Google Scholar,19Chevalier R.L. Forbes M.S. Thornhill B.A. Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.Kidney Int. 2009; 75: 1145-1152Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar,20Becknell B. Carpenter A.R. Allen J.L. Wilhide M.E. Ingraham S.E. Hains D.S. McHugh K.M. Molecular basis of renal adaptation in a murine model of congenital obstructive nephropathy.PLoS ONE. 2013; 8: e72762Crossref PubMed Scopus (16) Google Scholar Indeed, individual B1402 underwent bilateral ureteral reimplantation, only to develop recurrent VUR and worsening hydronephrosis, likely because her underlying bladder dysfunction had not been recognized. We propose that disruption of CHRNA3 can result in a pathophysiological sequence by which impaired neuronal innervation leads to bladder dysfunction, functional lower urinary tract obstruction, and subsequent upper urinary tract anomalies. In addition to their renal manifestations, families B1717 and GM, in whom truncating CHRNA3 mutations were found, have dysautonomic features, most notably an impaired pupillary light reflex. Compellingly, autoantibodies to the α3 nAChR subunit have been described to cause an autoimmune autonomic ganglionopathy in humans.21Vernino S. Sandroni P. Singer W. Low P.A. Invited Article: Autonomic ganglia: target and novel therapeutic tool.Neurology. 2008; 70: 1926-1932Crossref PubMed Scopus (93) Google Scholar These individuals develop profound autonomic failure, with symptoms overlapping those found in families with truncating CHRNA3 mutations, including bladder dysfunction, impaired pupillary light reflexes, and orthostatic hypotension.21Vernino S. Sandroni P. Singer W. Low P.A. Invited Article: Autonomic ganglia: target and novel therapeutic tool.Neurology. 2008; 70: 1926-1932Crossref PubMed Scopus (93) Google Scholar,22Vernino S. Low P.A. Fealey R.D. Stewart J.D. Farrugia G. Lennon V.A. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies.N. Engl. J. Med. 2000; 343: 847-855Crossref PubMed Scopus (508) Google Scholar Notably, individual B1402 does not have dysautonomic manifestations. This may be due to subtle findings that are not clinically manifest, or it may be the case that hypomorphic mutations lead to a milder phenotype. Identification of additional affected individuals with CHRNA3 mutations may provide further insight into genotype-phenotype correlations. The genitourinary and ocular phenotypes seen in families B1717 and GM are strikingly similar to that of the Chrna3−/− mice, which have megacystis, recurrent urinary tract infections, and persistent mydriasis.10Xu W. Gelber S. Orr-Urtreger A. Armstrong D. Lewis R.A. Ou C.N. Patrick J. Role L. De Biasi M. Beaudet A.L. Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 5746-5751Crossref PubMed Scopus (238) Google Scholar Bladder strips from these animals fail to contract in response to nicotine, and neurons from the superior cervical ganglia do not generate current in response to acetylcholine, consistent with the notion that loss of CHRNA3 results in impaired fast synaptic transmission within the autonomic ganglia.10Xu W. Gelber S. Orr-Urtreger A. Armstrong D. Lewis R.A. Ou C.N. Patrick J. Role L. De Biasi M. Beaudet A.L. Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 5746-5751Crossref PubMed Scopus (238) Google Scholar It is interesting to note that there is variable expressivity among affected individuals with CHRNA3 mutations. The two siblings in family B1717, who harbor the same CHRNA3 p.Thr337Asnfs∗87 truncating mutation, for example, exhibit a range of renal phenotypes, from only recurrent urinary tract infections to severe hydronephrosis, vesicoureteral reflux, and chronic kidney disease. This supports the notion that the renal disease manifesting in these individuals is the result of a pathophysiological sequence whereby impairment of bladder contraction results in secondary upper urinary tract malformations. The degree of renal impairment is likely a result of stochastic changes that occur in utero, and such variable expressivity is often seen in a variety of other monogenic diseases that cause CAKUT.4van der Ven A.T. Vivante A. Hildebrandt F. Novel Insights into the Pathogenesis of Monogenic Congenital Anomalies of the Kidney and Urinary Tract.J. Am. Soc. Nephrol. 2018; 29: 36-50Crossref PubMed Scopus (47) Google Scholar,23Vivante A. Hildebrandt F. 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Genet. 2011; 89: 668-674Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The findings in our study provide additional evidence that disruption of the neural pathways regulating bladder function can be important genetic causes of both CAKUT and autonomic dysfunction in humans. Our findings may point to important therapeutic implications. Current management for children with lower urinary obstruction involves surgical intervention to relieve anatomic obstruction and subsequent medical management of the sequelae from chronic kidney disease.31Chevalier R.L. Congenital urinary tract obstruction: the long view.Adv. Chronic Kidney Dis. 2015; 22: 312-319Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar However, surgical techniques alone may not be successful for individuals in whom mutations in CHRNA3 are identified, as was the case for individual B1402. The neuronal pathways regulating bladder contraction additionally provide tractable therapeutic targets that may be amenable to pharmacological intervention. In addition, early prenatal genetic diagnoses might eventually allow for pharmacological interventions in utero, which could prevent the development of renal dysgenesis. Further identification of novel genetic causes of urinary tract obstruction will provide additional strategies toward precision medicine. F.H. is a cofounder and S.A.C. member and holds stocks in Goldfinch-Bio. All other authors declare that they have no competing financial interests. We thank the affected individuals and their families for their contributions to this study. We also would like to thank Michael Wangler, Arthur Beaudet, Reza Bekheirnia, William Newman, and Fowzan Alkuraya for helpful discussion. This research was supported by grants from the National Institutes of Health to F.H. ( DK0668306 ). N.M. is supported by funding from the National Institutes of Health (grant T32-DK007726 ). F.K. is supported by funding from the Biomedical Education Program . D.M.C. is funded by the Health Research Board, Ireland ( HPF-206-674 ), the International Pediatric Research Foundation Early Investigators' Exchange Program , and the Amgen Irish Nephrology Society Specialist Registrar Bursary . M.N. is supported by a grant from the Japan Society for the Promotion of Science . V.K. is supported by the Deutsche Forschungsgemeinschaft ( 403877094 ). A.J.M. is supported by funding from the National Institutes of Health (grant T32-DK007726 ), the 2017 Harvard Stem Cell Institute Fellowship Grant , and the 2018 Jared J. Grantham Research Fellowship from the American Society of Nephrology Ben J. Lipps Research Fellowship Program. C.-H.W.W. is supported by funding from the National Institutes of Health (grant T32-GM007748 ). R.S.L. is supported by funding from the National Institutes of Health ( DK096238 ). S.E. is a Wellcome Senior Investigator. F.H. is also supported by the Begg Family Foundation. We also thank the Yale Center for Mendelian Genomics for whole-exome sequencing analysis ( U54HG006504 ). Download .pdf (1.09 MB) Help with pdf files Document S1. Supplemental Material and Methods, Figures S1–S5, and Table S1 1000 Genomes Project, https://www.internationalgenome.org/1000-genomes-browsersClustal Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/Exome Aggregation Consortium (ExAC), http://exac.broadinstitute.orgGenBank, https://www.ncbi.nlm.nih.gov/genbank/Genome Aggregation Database (gnomAD), http://gnomad.broadinstitute.orgMutationTaster, http://www.mutationtaster.orgNHLBI Exome Sequencing Project (ESP), https://evs.gs.washington.edu/EVSOnline Mendelian Inheritance in Man (OMIM), https://www.omim.orgPolyPhen2, http://genetics.bwh.harvard.edu/pph2Sorting Intolerant From Tolerant (SIFT), http://sift.jcvi.orgUCSC Genome Browser, https://genome.ucsc.edu